The NKG2D-Activating Receptor Mediates Pulmonary Clearance of Pseudomonas aeruginosa
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感染与免疫杂志 2006年第5期
Department of Environmental Health, Division of Environmental Genetics and Molecular Toxicology
Department of Internal Medicine, Division of Pulmonary and Critical Care, University of Cincinnati College of Medicine
Division of Pulmonary Medicine, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45267
ABSTRACT
The NKG2D-activating receptor is expressed on cytotoxic lymphocytes and interacts with ligands expressed on the surface of cells stressed by pathogenic and nonpathogenic stimuli. In this study, we investigated the physiologic importance of NKG2D receptor-ligand interactions in response to acute pulmonary Pseudomonas aeruginosa infection. P. aeruginosa infection increased the expression of mouse NKG2D ligands (Rae1) in airway epithelial cells and alveolar macrophages in vivo and also increased the cell surface expression of human NKG2D ligands (ULBP2) on airway epithelial cells in vitro. NKG2D receptor blockade with a specific monoclonal antibody inhibited the pulmonary clearance of P. aeruginosa. NKG2D receptor blockade also resulted in decreased production of Th1 cytokines and nitric oxide in the lungs of P. aeruginosa-infected mice. Additionally, NKG2D receptor blockade reduced the epithelial cell sloughing that accompanies P. aeruginosa infection. Macrophage phagocytosis and bronchoalveolar lavage cellularity were not different in P. aeruginosa-infected mice with and without NKG2D receptor blockade. These results demonstrate the importance of NKG2D-mediated immune activation in the clearance of acute bacterial infection and suggest that epithelial cell-lymphocyte interactions mediate pulmonary cytokine production, epithelial cell integrity, and bacterial clearance.
INTRODUCTION
Pseudomonas aeruginosa remains a major cause of nosocomial infections in hospitalized patients and immunocompromised individuals and is the predominant cause of morbidity and mortality in patients with cystic fibrosis (46). Important roles in pulmonary host defense against P. aeruginosa have been defined for many cell types, including respiratory epithelium, macrophages, and neutrophils. More recently, lymphocytes were demonstrated to play a critical role in the pulmonary defense against acute P. aeruginosa infection. Specifically, infection of lymphocyte-deficient (Rag2–/–) mice with P. aeruginosa leads to high mortality compared to the mortality of wild-type mice (39). The mechanisms and mediators involved in lymphocyte regulation of pulmonary P. aeruginosa clearance have not been established.
One potential mechanism may involve the recognition of infected or stressed airway epithelial cells by pulmonary lymphocytes. The immune system is capable of surveying infected or stressed tissue through the recognition of inhibitory and activating ligands recognized by cytotoxic T cells and natural killer (NK) cells. The immunosurveillance provided by these pathways is thought to facilitate the turnover of damaged or stressed host cells to control inflammation and promote epithelial repair. Multiple mechanisms for detection and elimination of stressed cells have been described (13, 36). One system that may provide a mechanistic link between cell stress and immune cell activation in the lung involves NKG2D receptor activation. The NKG2D receptor is expressed on circulating and tissue lymphocytes, predominantly NK cells, NK T cells, CD8+ T cells, and T cells (3, 11, 23). This receptor directly recognizes transformed or infected cells through structurally related ligands expressed on the surface of stressed cells (3, 45, 51). The proposed role of the NKG2D receptor in innate immune responses to cellular and tissue stress is based on the ability of the receptor to stimulate cytotoxic effects of NK cells and T cells against virally infected cells and tumor cells in vitro and in vivo (6). In addition to cytotoxicity, the recognition of NKG2D ligands induces production of several cytokines, including tumor necrosis factor alpha (TNF-) and gamma interferon (IFN-) (28).
Two families of NKG2D ligands have been identified in humans: the major histocompatibility complex class I chain-related molecules MICA and MICB (3) and the UL-16 binding proteins (ULBP) (2, 7, 24, 43). Several families of mouse NKG2D ligands have also been identified, including retinoic acid-inducible early (RAE-1) to RAE-1, H60, Mult1 (45), and Mill1 (26). NKG2D ligand expression is restricted or absent in normal tissues but is induced in response to various stresses and in some pathological conditions (17, 25). For example, NKG2D ligands are detected on human intestinal epithelial cells infected with Mycobacterium tuberculosis or Escherichia coli (48), and mouse NKG2D ligands are upregulated on the surface of peritoneal macrophages in response to Toll-like receptor (TLR) activation (19). Currently, there is no evidence that pathogenic stress can upregulate the cell surface expression of NKG2D ligands in the airways or that the expression of these ligands is physiologically significant. In the present study, we examined the expression of NKG2D ligands in the lung and investigated the role of the NKG2D receptor in the host defense against acute pulmonary P. aeruginosa infection.
MATERIALS AND METHODS
Mice. Six-week-old adult CD-1 mice were obtained from Charles River Laboratories (Wilmington, MA). All animal studies were carried out in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of Cincinnati College of Medicine.
Bacterial strains and growth conditions. Shaking cultures of P. aeruginosa strain PAO1 (21) and the PAO1 strain harboring a green fluorescent protein-expressing plasmid (PAO1-GFP) were grown in Luria broth (LB) at 37°C. When necessary, LB was supplemented with 1.5% Bacto agar. Carbenicillin (300 μg/ml) selection was performed for PAO1-GFP whenever it was appropriate.
Mouse acute lung infection and bacterial enumeration. Mice were infected intranasally with 106 CFU of stationary-phase P. aeruginosa strain PAO1. The lungs of infected mice were harvested and homogenized, serial dilutions of lung homogenates were plated onto LB agar plates, and the CFU were enumerated as previously described (30).
RAE-1 immunohistochemistry. Mice were anesthetized (50 mg/kg of pentobarbital sodium given intraperitoneally) and exsanguinated by severing the posterior abdominal aorta. To obtain tissue for histological analysis, a cannula was inserted into the trachea, and the lungs were instilled with 10% phosphate-buffered formalin at a constant pressure (25 cm H2O). The trachea was ligated, and the inflated lungs were immersed in fixative for 24 h. An RAE-1 immunohistochemistry analysis was performed with a goat polyclonal antibody raised against RAE-1 (clone AF1136; R&D Systems, Minneapolis, MN). This antibody also recognizes RAE-1, -, -, and -. Antigen-antibody complexes were detected in paraffin-embedded tissue sections (5 μm) of mouse lungs (four or five sections per group) with a Vectastain ABC Elite immunoglobulin G (IgG) kit (Vector Laboratories, Burlingame, CA) used according to the manufacturer's protocol.
Lung cell isolation and flow cytometry. Mice were anesthetized as described above, and lungs were perfused with 10 ml phosphate-buffered saline (PBS) containing 0.6 mM EDTA, removed, and diced to obtain pieces whose volume was <300 μl. Four milliliters of Hanks' balanced salt solution (HBSS) containing 175 U/ml collagenase, 0.2 U/ml pancreatic elastase, 35 U/ml hyaluronidase, 20 kU/ml DNase (Sigma, St. Louis, MO), 10% fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin was added to the tissue and incubated for 30 min at 37°C in an orbital shaker. The digested lungs were sheared with a 20-gauge needle and filtered through 40-μm filters. CD8+ cells (clone 53-6.7; Pharmingen, San Diego, CA), natural killer cells (NK1.1; clone PK136; eBioscience, San Diego, CA), and NKG2D receptor (rat monoclonal anti-mouse NKG2D antibody; clone 191004; R&D Systems)-positive cell populations were identified by flow cytometry. Flow cytometry was performed with a FACScan cytofluorometer (Becton Dickinson, Franklin Lakes, NJ), and data acquisition and analysis were performed using the CellQuest software (Becton Dickinson).
Human NKG2D ligand expression. Immortalized human tracheal epithelial cells (9HTEo- cells) were purchased from the American Type Culture Collection (Manassas, VA) and were maintained in Dulbecco modified Eagle medium (MediaTech, Herndon, VA) supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO), 100 U/ml penicillin, and 100 mg/ml streptomycin (Sigma). The cells were maintained at 37°C in a humidified incubator containing 5% CO2-95% air. For P. aeruginosa exposure, cells were washed twice with PBS and incubated with P. aeruginosa (multiplicity of infection, 10) in PBS for 2 h at 37°C. Following incubation, cells were washed twice, replenished with Dulbecco modified Eagle medium, and incubated for 24 h at 37°C in a humidified incubator containing 5% CO2-95% air. Cells used for flow cytometry were harvested in 2 ml of nonenzymatic cell dissociation solution (10 mM EDTA in PBS without Ca2+ and Mg2+). The cells were washed in 0.5% bovine serum albumin-0.05% sodium azide in PBS (FACS buffer). For cell surface staining of ULBP2 ligands, cells were resuspended in 100 μl FACS buffer and incubated with 10 μg/ml of primary antibodies for 60 min at 4°C. The primary mouse monoclonal IgG antibodies used were anti-ULBP2 (clone AF1298; R&D Systems) and isotype control mouse IgG (clone MOPC-173; BD Pharmingen). Cells were washed and resuspended in 100 μl of FACS buffer. Cells were then incubated with 20 μg/ml phycoerythrin-conjugated goat anti-mouse IgG secondary antibody (BD Biosciences, San Jose, CA) for 30 min at 4°C. Cells were washed and fixed in 400 μl of 2.0% paraformaldehyde (pH 7.2). Flow cytometry was performed using a Beckman-Coulter flow cytometer (Epics XL-MCL, Fullerton, CA), and the data were analyzed using the WinList 5.0 software (Verity, Topsham, ME).
Western blot analyses. The protein used for Western blot analysis was isolated using the mammalian protein extraction reagent (Pierce, Rockford, IL) according to the manufacturer's instructions. Supernatants were aliquoted for storage at –80°C, and protein was quantitated using a bicinchoninic acid assay (Pierce). Western blot protein samples (40 μg) were loaded into a NuPAGE 4 to 12% bis-Tris gel (Invitrogen) and electrophoresed using the protocol for an XCell Surelock Mini-cell in NuPAGE morpholineethanesulfonic acid (MES)-sodium dodecyl sulfate running buffer (Invitrogen). Following electrophoresis, the gels were transferred onto polyvinylidene difluoride membranes. The membranes were incubated overnight with anti-RAE-1 (1-μg/ml dilution; clone AF1136; R&D Systems, Minneapolis, MN). The membranes were then incubated with bovine anti-goat IgG-horseradish peroxidase-conjugated secondary antibody, and detection was performed using ECL reagents (Amersham, Piscataway, NJ). The membranes were exposed to Biomax XAR film (Kodak, Rochester, NY). Equal protein loading was verified by staining the gels with Gel Code Blue (Pierce).
Anti-NKG2D administration. The NKG2D receptor function was blocked by administration of the rat anti-mouse NKG2D monoclonal antibody CX5 under conditions identical to those described previously (41). The CX5 antibody blocks binding of NKG2D to its ligands, RAE-1 and H60, and inhibits NKG2D-dependent NK cell-mediated cytotoxicity against NKG2D ligand-bearing cells in vitro and in vivo (40, 41). Briefly, 12 to 16 h prior to infection, mice were inoculated intraperitoneally with either 100 μg rat anti-mouse NKG2D monoclonal antibody CX5 (eBioscience) or 100 μg control rat IgG. Injection of anti-mouse NKG2D monoclonal antibody CX5 did not deplete NKG2D-expressing cells because the frequencies of NK cells (as determined by NK cell-specific staining; clone DX5; eBioscience) and CD8+ cells were not reduced compared to the frequencies observed for control mice inoculated with IgG.
BAL and cell enumeration. Mice were anesthetized (50 mg/kg of pentobarbital sodium given intraperitoneally) and exsanguinated by severing the posterior abdominal aorta. The lungs were then lavaged two times with 1 ml of HBSS. Individual bronchoalveolar lavage (BAL) returns were pooled and centrifuged at 300 x g for 10 min. The supernatant was removed and stored at –70°C until it was assayed. The cell pellet was reconstituted in 1 ml HBSS containing 2% fetal bovine serum. Total cell counts were determined with a hemocytometer. Differential leukocyte counts (>300 cells) were determined on Hemacolor-stained (EM Science, Gibbstown, NJ) cytospin slides (Cytospin3; Shandon Scientific Ltd., Astmoor, Runcorn, England). Airway epithelial cells were enumerated on the same slides, and the results were expressed as percentages of the total number of cells in the BAL fluid.
Enzyme-linked immunosorbent assay. Mice were anesthetized as described above and were exsanguinated by severing the posterior abdominal aorta. The lungs were removed, immersed in liquid nitrogen, and homogenized in PBS. Cytokine protein levels in lung homogenates were determined by an enzyme-linked immunosorbent assay performed according to the manufacturer's protocols (BD Biosciences, Carlsbad, CA).
Macrophage phagocytosis. In vivo macrophage phagocytosis was assessed as described previously (33). CD-1 mice (groups of four to six mice) pretreated with neutralizing NKG2D antibody or with control IgG were infected intranasally with 1 x 107 PAO1-GFP cells. At 8 and 24 h after bacterial infection, mouse lungs were lavaged with three 1-ml aliquots of PBS. The resulting BAL fluid was pooled and centrifuged (400 x g, 4°C). The cell pellet was washed three times in PBS to separate the uningested, extracellular bacteria from BAL cells. The resulting cells were fixed in 1% formalin buffered with PBS. The percentage of macrophages with engulfed bacteria was determined with a phase-contrast fluorescence microscope. At least 200 macrophages were counted in BAL fluid from each mouse. Macrophages were identified morphologically at high magnification. Extracellular bacteria were quenched with crystal violet (0.8 mg/ml).
Detection of nitrite levels. Nitric oxide (NO) levels in the BAL fluid were estimated by the measuring nitrite levels using the Griess reagent system according to the manufacturer's instructions (Promega, Madison, WI). Briefly, 50 μl of BAL fluid was incubated with 50 μl of Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamide in 5% phosphoric acid) in a 96-well tissue culture plate. The optical density at 550 nm was determined with a Spectramax microplate reader (Molecular Devices, Sunnyvale, CA). Nitrite concentrations were calculated by comparison with the optical densities at 550 nm of standard solutions of sodium nitrite (0 to 100 μM).
Statistics. Parametric data were analyzed for statistical significance by a one-way analysis of variance, followed by Student t tests. Differences between means were considered significant when the P value was <0.05.
RESULTS
Expression and induction of NKG2D ligands during P. aeruginosa infection. P. aeruginosa infection markedly increased the expression of the RAE-1 family of NKG2D ligands in whole lungs, as determined by Western blot analysis. An 37-kDa band was detected following N-glyconase treatment in uninfected mice, and the band intensity increased in a time-dependent manner (Fig. 1A) over a 24-h period. An immunohistochemistry analysis was performed to further examine the cellular sources of RAE-1. Minimal staining was evident in the conducting airways, alveolar epithelium, or alveolar macrophages (AM) of uninfected mice (Fig. 1B and D). In contrast, RAE-1-specific staining revealed that the conducting airway epithelium, alveolar epithelium, and alveolar macrophages were strongly positive 24 h after P. aeruginosa infection (Fig. 1C and E).
P. aeruginosa directly stimulates NKG2D ligand expression on human airway epithelial cells. The ability of P. aeruginosa to directly stimulate NKG2D ligands was examined in vitro using the simian virus 40-transformed human tracheal epithelial cell line 9HTEo-. Consistent with airway epithelial cell RAE-1 expression in vivo, minimal ULBP2 ligand expression was detected on uninfected 9HTEo- cells in vitro. Analogous to the in vivo model, expression of the human NKG2D ligand ULBP2 increased following 24 h of P. aeruginosa infection in vitro (Fig. 2).
Cells expressing NKG2D receptor are resident cells of the lung. NK cells, CD8+ T cells, and + T cells are known to express NKG2D in mice (12, 23). Lung digestion and flow cytometry were performed to assess the presence of lymphocytes bearing the NKG2D receptor in the lungs of nave mice. As determined with a rat monoclonal antibody specific for mouse NKG2D, 20% of the lymphocyte population obtained from the perfused lungs expressed NKG2D (Fig. 3A and B), indicating that a significant population of receptor-bearing cells resides in the lung. The majority of the NK1.1+ cells (82%), but only 12% of the CD8+ cells, coexpressed the NKG2D receptor in the lung (Fig. 3C and D). There were no significant differences in the distribution of NKG2D-positive cells in the lung 24 h after P. aeruginosa infection (not shown).
NKG2D receptor blockade inhibits bacterial clearance in the lung. To examine the function of the NKG2D receptor-ligand system in acute bacterial infection, we blocked NKG2D function in vivo by administration of the CX5 blocking antibody 16 h prior to P. aeruginosa infection. NKG2D blockade significantly impaired pulmonary clearance 24 h after P. aeruginosa infection (Fig. 4). Mice pretreated with the anti-NKG2D antibody had approximately 20-fold more bacilli in their lungs than control PAO1-infected mice had. These results directly implicate NKG2D receptor activation in the host response to P. aeruginosa because NKG2D cells were not depleted by antibody administration, as the frequencies of NK cells and CD8+ cells were not reduced compared to the frequencies in control mice inoculated with IgG (not shown).
Inhibition of bacterial clearance with NKG2D receptor blockade is independent of macrophage phagocytosis. Given the importance of AM phagocytosis in bacterial clearance, we next examined whether inhibition of AM phagocytosis accounted for the decreased bacterial clearance in mice given the NKG2D blocking antibody. The number of BAL-derived AM that had ingested bacteria was slightly greater in mice that received the NKG2D receptor blockade than in control mice at 2 h postinfection (Fig. 5). There were no differences in AM phagocytosis at 8 h or 24 h.
NKG2D receptor blockade inhibits pulmonary cytokine production, NO production, and epithelial cell damage. Following 24 h of P. aeruginosa infection, we examined the expression of pleiotropic inflammatory cytokines (interleukin-1 [IL-1], TNF-, and IFN-), leukocyte inflammation, epithelial cell shedding, and bacterial clearance from the lung. Anti-NKG2D pretreatment significantly decreased the pulmonary cytokine levels of IL-1, TNF-, and IFN- at 24 h postinfection (Fig. 6A to C). Similarly, NO levels, as estimated from the nitrite concentration, were reduced in the BAL fluid of mice that had been pretreated with anti-NKG2D antibody (Fig. 7). Reduced inflammatory cytokine levels were not accompanied by significant changes in the inflammatory cell profile in the BAL fluid (Fig. 8A). There was also no significant difference in the total leukocyte counts between treatments at 24 h (control, 2.68 x 106 ± 0.36 x 106 cells; anti-NKG2D treatment, 2.21 x 106 ± 0.26 x 106 cells). Although there were no differences in the leukocyte number and composition in the BAL fluid between treatments, there was a significant decrease in airway epithelial cell shedding in mice pretreated with anti-NKG2D compared to the shedding in control-treated mice. Approximately three times more epithelial cell damage occurred in the IgG-treated P. aeruginosa-infected mice, as determined by the number of epithelial cells recovered in the BAL fluid (Fig. 8B).
DISCUSSION
Although airway epithelial cells are the first line of defense against environmental pathogens, our knowledge of the role of these cells in signaling to resident mucosal immune cells is limited. Our results provide evidence which further establishes that epithelial cell-lymphocyte interactions mediate pulmonary cytokine production, epithelial cell integrity, and bacterial clearance. Specifically, our data demonstrate that the NKG2D receptor function is an important mechanism in the host defense against acute P. aeruginosa infection.
NKG2D ligand expression represents an intriguing link between the host encounter with a pathogen and subsequent activation of the immune system. The NKG2D receptor recognizes stressed cells through structurally related ligands induced on the cell surface (45). The NKG2D receptor was first detected on natural killer cells (52), and its function was subsequently determined to be an activating receptor in response to major histocompatibility complex class I-like molecules upregulated in innate and adaptive immune responses to cellular and tissue stress (3, 6, 51). Subsequent investigations detected NKG2D on NK T cells, CD8+ T cells, and T cells (45). Our finding that RAE-1 ligands are upregulated in the lung in response to P. aeruginosa infection is consistent with the role proposed for these molecules in tissue surveillance. Immunoblot and anti-RAE-1 immunohistochemistry analyses provide direct in vivo evidence that lung epithelial cells rapidly respond to pathogenic stimuli and are capable of directly activating the pulmonary immune system. Furthermore, incubation of human airway epithelial cells with P. aeruginosa directly stimulated induction of the human NKG2D ligand ULBP2 (Fig. 2). This is significant because it suggests that direct recognition of the pathogen, possibly via TLR binding, is sufficient to induce NKG2D ligands. Along these lines, various strains of E. coli induce expression of the NKG2D ligand MICA on Caco-2 human intestinal epithelial cells in vitro (48). In addition, Hamerman et al. recently demonstrated that RAE-1 transcript and cell surface protein induction occurs in peritoneal macrophages in response to TLR ligands, including lipopolysaccharide, zymosan, and double-stranded RNA (19).
This study provides the first demonstration that the NKG2D effector function is required for a competent host response against a pathogen in vivo. The cellular and molecular events that mediate the progression of bacterial infection via NKG2D activation are not entirely clear, but they may involve attenuated production of cytokines by NKG2D-expressing cells in the lung. A key feature of the NKG2D receptor-ligand system is that the function does not depend on an increase in the number of effector cells but does depend on the condition of the tissue and its capacity to stimulate resident immune cells. Significant populations of NKG2D-expressing cells (i.e., NK cells, CD8+ cells) reside in the pulmonary submucosa and parenchyma of unexposed mice (Fig. 3), and resident lymphocytes in the lung are prodigious sources of cytokines, including IFN-, TNF-, and IL-1 implicated in the host response to bacterial pathogens (5, 15, 18, 31, 32). There are conflicting data on the significance of proinflammatory cytokines in the context of pulmonary infection. Several studies have demonstrated that IFN- production by NK T cells is beneficial in the host response to microbial infections, including P. aeruginosa infections (14, 27, 29, 39). Furthermore, Wiemann et al. recently reported that systemic downregulation of NKG2D impairs the ability of NK cells and CD8+ T cells to produce IFN- in response to Listeria monocytogenes infection (50). In contrast, other investigators have shown that IFN- is dispensable for effective clearance of a P. aeruginosa infection (38, 47). We predict that the inhibition of cytokine production in mice that receive NKG2D blockade represents a proximal event in the subsequent cascade of cytokine production and inhibits macrophage and epithelial cell functions (e.g., direct microbicidal activity) in response to P. aeruginosa infection. The central role of activated macrophages and airway epithelium in the clearance of P. aeruginosa is supported by the fact that neutrophil accumulation is not affected in mice that receive NKG2D blocking antibody compared to neutrophil accumulation in control mice. The data presented here support the idea that the reduction in proinflammatory or activating cytokines may contribute to the immunosuppression observed in NKG2D antibody-treated mice. Furthermore, the inhibition of cytokine production may reflect a generalized depression in the lymphocyte-mediated antibacterial defense mechanisms. However, without conclusive evidence for a mechanism(s) responsible for the reduced clearance, the data should be interpreted cautiously.
Two key components of antibacterial defense in the lung, neutrophil accumulation and macrophage phagocytosis, are not affected by NKG2D blockade at 24 h and likely do not account for the mechanism by which NKG2D activation mediates the progression of bacterial growth. The lack of effects on inflammation are not totally surprising given that studies have shown that the cytokines are not necessary for increased inflammation in animal models (4, 15). The fact that phagocytosis is not impaired is somewhat surprising given that IFN- is a key mediator in the activation of alveolar macrophage functions and the elaboration of oxidative metabolites (8-10), which are compromised in anti-NKG2D-treated mice (Fig. 7). We speculate that additional components of the antibacterial defense repertoire of the lung may also be compromised as a consequence of NKG2D receptor blockade. These pathways include elaboration of antimicrobial peptides from the airway epithelium and the possibility that NKG2D-expressing cells may directly contribute to the antimicrobial shield in the lung. Human pulmonary epithelial cells are capable of producing -defensin antibacterial peptides in response to P. aeruginosa infection, as well as TNF- and IL-1 (20), and mice deficient in -defensin1 exhibit compromised clearance of several pulmonary pathogens (37). Furthermore, IL-1 and TNF- production by lipopolysaccharide-stimulated monocytes is sufficient to increase the expression of -defensin2 in human pulmonary epithelial cells (49). Another facet of NKG2D activation that remains to be explored is the potential production of antimicrobial peptides by T cells and NK cells in response to NKG2D ligand expression. T cells and NK cells are known to produce factors that are directly cytotoxic to bacteria (1, 34, 35, 42), but the relevance and significance of these mediators remain to be elucidated in our system.
There is conflicting evidence concerning the consequences of epithelial cell injury in the context of bacterial clearance. Our data demonstrate that increased epithelial cell death is associated with increased bacterial clearance and are in agreement with one of the known functions of the NKG2D receptor-ligand system (i.e., cytotoxic effects against ligand-bearing cells). In this regard, Grassme et al. demonstrated that bronchial cell apoptosis does not occur in P. aeruginosa-infected mice deficient in the CD95 cell death receptor, which is accompanied by increased mortality compared to the mortality of wild-type infected mice (16). This indicates that shedding of airway epithelial cells in control mice prevents bacterial dissemination and thus contributes to bacterial clearance. In contrast, airway epithelial cells in culture are relatively resistant to P. aeruginosa-induced apoptosis (44), and nominal apoptosis of airway epithelium was reported in septic patients (22). These studies and our study should be interpreted cautiously in this regard, because these reports provide data obtained with different strains of pathogens, different experimental designs, and different reagents used to detect apoptosis.
The present study revealed that the induction of RAE-1 ligands and subsequent immune activation through the NKG2D receptor are a significant component of the pulmonary host defense against acute P. aeruginosa infection. This pathway does not involve significant alterations in inflammation or AM phagocytosis, but it does regulate the elaboration of key cytokines involved in host defense, including IL-1, TNF-, and IFN-. We hypothesize that NKG2D activation is a key regulator of cytokine production by pulmonary T cells and NK cells and that inhibition of this production by NKG2D blockade leads to suppression of subsequent mediators of host defense. Further investigation of the mechanism of NKG2D receptor-ligand interactions in the lung should contribute to our understanding of the pulmonary innate immune system. Therapeutic strategies based solely on antibiotics remain limited due to the extensive ability of P. aeruginosa to develop antibiotic resistance, and novel strategies that enhance the host immune response against this pathogen may lead to reagents that improve the outcome of treatment of P. aeruginosa infections.
ACKNOWLEDGMENTS
We thank Michael P. Davis and Steve Ziegler for helpful discussions.
This study was supported by the NIH/NIEHS Center for Environmental Genetics (grant P30-ES06096-02).
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Department of Internal Medicine, Division of Pulmonary and Critical Care, University of Cincinnati College of Medicine
Division of Pulmonary Medicine, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45267
ABSTRACT
The NKG2D-activating receptor is expressed on cytotoxic lymphocytes and interacts with ligands expressed on the surface of cells stressed by pathogenic and nonpathogenic stimuli. In this study, we investigated the physiologic importance of NKG2D receptor-ligand interactions in response to acute pulmonary Pseudomonas aeruginosa infection. P. aeruginosa infection increased the expression of mouse NKG2D ligands (Rae1) in airway epithelial cells and alveolar macrophages in vivo and also increased the cell surface expression of human NKG2D ligands (ULBP2) on airway epithelial cells in vitro. NKG2D receptor blockade with a specific monoclonal antibody inhibited the pulmonary clearance of P. aeruginosa. NKG2D receptor blockade also resulted in decreased production of Th1 cytokines and nitric oxide in the lungs of P. aeruginosa-infected mice. Additionally, NKG2D receptor blockade reduced the epithelial cell sloughing that accompanies P. aeruginosa infection. Macrophage phagocytosis and bronchoalveolar lavage cellularity were not different in P. aeruginosa-infected mice with and without NKG2D receptor blockade. These results demonstrate the importance of NKG2D-mediated immune activation in the clearance of acute bacterial infection and suggest that epithelial cell-lymphocyte interactions mediate pulmonary cytokine production, epithelial cell integrity, and bacterial clearance.
INTRODUCTION
Pseudomonas aeruginosa remains a major cause of nosocomial infections in hospitalized patients and immunocompromised individuals and is the predominant cause of morbidity and mortality in patients with cystic fibrosis (46). Important roles in pulmonary host defense against P. aeruginosa have been defined for many cell types, including respiratory epithelium, macrophages, and neutrophils. More recently, lymphocytes were demonstrated to play a critical role in the pulmonary defense against acute P. aeruginosa infection. Specifically, infection of lymphocyte-deficient (Rag2–/–) mice with P. aeruginosa leads to high mortality compared to the mortality of wild-type mice (39). The mechanisms and mediators involved in lymphocyte regulation of pulmonary P. aeruginosa clearance have not been established.
One potential mechanism may involve the recognition of infected or stressed airway epithelial cells by pulmonary lymphocytes. The immune system is capable of surveying infected or stressed tissue through the recognition of inhibitory and activating ligands recognized by cytotoxic T cells and natural killer (NK) cells. The immunosurveillance provided by these pathways is thought to facilitate the turnover of damaged or stressed host cells to control inflammation and promote epithelial repair. Multiple mechanisms for detection and elimination of stressed cells have been described (13, 36). One system that may provide a mechanistic link between cell stress and immune cell activation in the lung involves NKG2D receptor activation. The NKG2D receptor is expressed on circulating and tissue lymphocytes, predominantly NK cells, NK T cells, CD8+ T cells, and T cells (3, 11, 23). This receptor directly recognizes transformed or infected cells through structurally related ligands expressed on the surface of stressed cells (3, 45, 51). The proposed role of the NKG2D receptor in innate immune responses to cellular and tissue stress is based on the ability of the receptor to stimulate cytotoxic effects of NK cells and T cells against virally infected cells and tumor cells in vitro and in vivo (6). In addition to cytotoxicity, the recognition of NKG2D ligands induces production of several cytokines, including tumor necrosis factor alpha (TNF-) and gamma interferon (IFN-) (28).
Two families of NKG2D ligands have been identified in humans: the major histocompatibility complex class I chain-related molecules MICA and MICB (3) and the UL-16 binding proteins (ULBP) (2, 7, 24, 43). Several families of mouse NKG2D ligands have also been identified, including retinoic acid-inducible early (RAE-1) to RAE-1, H60, Mult1 (45), and Mill1 (26). NKG2D ligand expression is restricted or absent in normal tissues but is induced in response to various stresses and in some pathological conditions (17, 25). For example, NKG2D ligands are detected on human intestinal epithelial cells infected with Mycobacterium tuberculosis or Escherichia coli (48), and mouse NKG2D ligands are upregulated on the surface of peritoneal macrophages in response to Toll-like receptor (TLR) activation (19). Currently, there is no evidence that pathogenic stress can upregulate the cell surface expression of NKG2D ligands in the airways or that the expression of these ligands is physiologically significant. In the present study, we examined the expression of NKG2D ligands in the lung and investigated the role of the NKG2D receptor in the host defense against acute pulmonary P. aeruginosa infection.
MATERIALS AND METHODS
Mice. Six-week-old adult CD-1 mice were obtained from Charles River Laboratories (Wilmington, MA). All animal studies were carried out in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of Cincinnati College of Medicine.
Bacterial strains and growth conditions. Shaking cultures of P. aeruginosa strain PAO1 (21) and the PAO1 strain harboring a green fluorescent protein-expressing plasmid (PAO1-GFP) were grown in Luria broth (LB) at 37°C. When necessary, LB was supplemented with 1.5% Bacto agar. Carbenicillin (300 μg/ml) selection was performed for PAO1-GFP whenever it was appropriate.
Mouse acute lung infection and bacterial enumeration. Mice were infected intranasally with 106 CFU of stationary-phase P. aeruginosa strain PAO1. The lungs of infected mice were harvested and homogenized, serial dilutions of lung homogenates were plated onto LB agar plates, and the CFU were enumerated as previously described (30).
RAE-1 immunohistochemistry. Mice were anesthetized (50 mg/kg of pentobarbital sodium given intraperitoneally) and exsanguinated by severing the posterior abdominal aorta. To obtain tissue for histological analysis, a cannula was inserted into the trachea, and the lungs were instilled with 10% phosphate-buffered formalin at a constant pressure (25 cm H2O). The trachea was ligated, and the inflated lungs were immersed in fixative for 24 h. An RAE-1 immunohistochemistry analysis was performed with a goat polyclonal antibody raised against RAE-1 (clone AF1136; R&D Systems, Minneapolis, MN). This antibody also recognizes RAE-1, -, -, and -. Antigen-antibody complexes were detected in paraffin-embedded tissue sections (5 μm) of mouse lungs (four or five sections per group) with a Vectastain ABC Elite immunoglobulin G (IgG) kit (Vector Laboratories, Burlingame, CA) used according to the manufacturer's protocol.
Lung cell isolation and flow cytometry. Mice were anesthetized as described above, and lungs were perfused with 10 ml phosphate-buffered saline (PBS) containing 0.6 mM EDTA, removed, and diced to obtain pieces whose volume was <300 μl. Four milliliters of Hanks' balanced salt solution (HBSS) containing 175 U/ml collagenase, 0.2 U/ml pancreatic elastase, 35 U/ml hyaluronidase, 20 kU/ml DNase (Sigma, St. Louis, MO), 10% fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin was added to the tissue and incubated for 30 min at 37°C in an orbital shaker. The digested lungs were sheared with a 20-gauge needle and filtered through 40-μm filters. CD8+ cells (clone 53-6.7; Pharmingen, San Diego, CA), natural killer cells (NK1.1; clone PK136; eBioscience, San Diego, CA), and NKG2D receptor (rat monoclonal anti-mouse NKG2D antibody; clone 191004; R&D Systems)-positive cell populations were identified by flow cytometry. Flow cytometry was performed with a FACScan cytofluorometer (Becton Dickinson, Franklin Lakes, NJ), and data acquisition and analysis were performed using the CellQuest software (Becton Dickinson).
Human NKG2D ligand expression. Immortalized human tracheal epithelial cells (9HTEo- cells) were purchased from the American Type Culture Collection (Manassas, VA) and were maintained in Dulbecco modified Eagle medium (MediaTech, Herndon, VA) supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO), 100 U/ml penicillin, and 100 mg/ml streptomycin (Sigma). The cells were maintained at 37°C in a humidified incubator containing 5% CO2-95% air. For P. aeruginosa exposure, cells were washed twice with PBS and incubated with P. aeruginosa (multiplicity of infection, 10) in PBS for 2 h at 37°C. Following incubation, cells were washed twice, replenished with Dulbecco modified Eagle medium, and incubated for 24 h at 37°C in a humidified incubator containing 5% CO2-95% air. Cells used for flow cytometry were harvested in 2 ml of nonenzymatic cell dissociation solution (10 mM EDTA in PBS without Ca2+ and Mg2+). The cells were washed in 0.5% bovine serum albumin-0.05% sodium azide in PBS (FACS buffer). For cell surface staining of ULBP2 ligands, cells were resuspended in 100 μl FACS buffer and incubated with 10 μg/ml of primary antibodies for 60 min at 4°C. The primary mouse monoclonal IgG antibodies used were anti-ULBP2 (clone AF1298; R&D Systems) and isotype control mouse IgG (clone MOPC-173; BD Pharmingen). Cells were washed and resuspended in 100 μl of FACS buffer. Cells were then incubated with 20 μg/ml phycoerythrin-conjugated goat anti-mouse IgG secondary antibody (BD Biosciences, San Jose, CA) for 30 min at 4°C. Cells were washed and fixed in 400 μl of 2.0% paraformaldehyde (pH 7.2). Flow cytometry was performed using a Beckman-Coulter flow cytometer (Epics XL-MCL, Fullerton, CA), and the data were analyzed using the WinList 5.0 software (Verity, Topsham, ME).
Western blot analyses. The protein used for Western blot analysis was isolated using the mammalian protein extraction reagent (Pierce, Rockford, IL) according to the manufacturer's instructions. Supernatants were aliquoted for storage at –80°C, and protein was quantitated using a bicinchoninic acid assay (Pierce). Western blot protein samples (40 μg) were loaded into a NuPAGE 4 to 12% bis-Tris gel (Invitrogen) and electrophoresed using the protocol for an XCell Surelock Mini-cell in NuPAGE morpholineethanesulfonic acid (MES)-sodium dodecyl sulfate running buffer (Invitrogen). Following electrophoresis, the gels were transferred onto polyvinylidene difluoride membranes. The membranes were incubated overnight with anti-RAE-1 (1-μg/ml dilution; clone AF1136; R&D Systems, Minneapolis, MN). The membranes were then incubated with bovine anti-goat IgG-horseradish peroxidase-conjugated secondary antibody, and detection was performed using ECL reagents (Amersham, Piscataway, NJ). The membranes were exposed to Biomax XAR film (Kodak, Rochester, NY). Equal protein loading was verified by staining the gels with Gel Code Blue (Pierce).
Anti-NKG2D administration. The NKG2D receptor function was blocked by administration of the rat anti-mouse NKG2D monoclonal antibody CX5 under conditions identical to those described previously (41). The CX5 antibody blocks binding of NKG2D to its ligands, RAE-1 and H60, and inhibits NKG2D-dependent NK cell-mediated cytotoxicity against NKG2D ligand-bearing cells in vitro and in vivo (40, 41). Briefly, 12 to 16 h prior to infection, mice were inoculated intraperitoneally with either 100 μg rat anti-mouse NKG2D monoclonal antibody CX5 (eBioscience) or 100 μg control rat IgG. Injection of anti-mouse NKG2D monoclonal antibody CX5 did not deplete NKG2D-expressing cells because the frequencies of NK cells (as determined by NK cell-specific staining; clone DX5; eBioscience) and CD8+ cells were not reduced compared to the frequencies observed for control mice inoculated with IgG.
BAL and cell enumeration. Mice were anesthetized (50 mg/kg of pentobarbital sodium given intraperitoneally) and exsanguinated by severing the posterior abdominal aorta. The lungs were then lavaged two times with 1 ml of HBSS. Individual bronchoalveolar lavage (BAL) returns were pooled and centrifuged at 300 x g for 10 min. The supernatant was removed and stored at –70°C until it was assayed. The cell pellet was reconstituted in 1 ml HBSS containing 2% fetal bovine serum. Total cell counts were determined with a hemocytometer. Differential leukocyte counts (>300 cells) were determined on Hemacolor-stained (EM Science, Gibbstown, NJ) cytospin slides (Cytospin3; Shandon Scientific Ltd., Astmoor, Runcorn, England). Airway epithelial cells were enumerated on the same slides, and the results were expressed as percentages of the total number of cells in the BAL fluid.
Enzyme-linked immunosorbent assay. Mice were anesthetized as described above and were exsanguinated by severing the posterior abdominal aorta. The lungs were removed, immersed in liquid nitrogen, and homogenized in PBS. Cytokine protein levels in lung homogenates were determined by an enzyme-linked immunosorbent assay performed according to the manufacturer's protocols (BD Biosciences, Carlsbad, CA).
Macrophage phagocytosis. In vivo macrophage phagocytosis was assessed as described previously (33). CD-1 mice (groups of four to six mice) pretreated with neutralizing NKG2D antibody or with control IgG were infected intranasally with 1 x 107 PAO1-GFP cells. At 8 and 24 h after bacterial infection, mouse lungs were lavaged with three 1-ml aliquots of PBS. The resulting BAL fluid was pooled and centrifuged (400 x g, 4°C). The cell pellet was washed three times in PBS to separate the uningested, extracellular bacteria from BAL cells. The resulting cells were fixed in 1% formalin buffered with PBS. The percentage of macrophages with engulfed bacteria was determined with a phase-contrast fluorescence microscope. At least 200 macrophages were counted in BAL fluid from each mouse. Macrophages were identified morphologically at high magnification. Extracellular bacteria were quenched with crystal violet (0.8 mg/ml).
Detection of nitrite levels. Nitric oxide (NO) levels in the BAL fluid were estimated by the measuring nitrite levels using the Griess reagent system according to the manufacturer's instructions (Promega, Madison, WI). Briefly, 50 μl of BAL fluid was incubated with 50 μl of Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamide in 5% phosphoric acid) in a 96-well tissue culture plate. The optical density at 550 nm was determined with a Spectramax microplate reader (Molecular Devices, Sunnyvale, CA). Nitrite concentrations were calculated by comparison with the optical densities at 550 nm of standard solutions of sodium nitrite (0 to 100 μM).
Statistics. Parametric data were analyzed for statistical significance by a one-way analysis of variance, followed by Student t tests. Differences between means were considered significant when the P value was <0.05.
RESULTS
Expression and induction of NKG2D ligands during P. aeruginosa infection. P. aeruginosa infection markedly increased the expression of the RAE-1 family of NKG2D ligands in whole lungs, as determined by Western blot analysis. An 37-kDa band was detected following N-glyconase treatment in uninfected mice, and the band intensity increased in a time-dependent manner (Fig. 1A) over a 24-h period. An immunohistochemistry analysis was performed to further examine the cellular sources of RAE-1. Minimal staining was evident in the conducting airways, alveolar epithelium, or alveolar macrophages (AM) of uninfected mice (Fig. 1B and D). In contrast, RAE-1-specific staining revealed that the conducting airway epithelium, alveolar epithelium, and alveolar macrophages were strongly positive 24 h after P. aeruginosa infection (Fig. 1C and E).
P. aeruginosa directly stimulates NKG2D ligand expression on human airway epithelial cells. The ability of P. aeruginosa to directly stimulate NKG2D ligands was examined in vitro using the simian virus 40-transformed human tracheal epithelial cell line 9HTEo-. Consistent with airway epithelial cell RAE-1 expression in vivo, minimal ULBP2 ligand expression was detected on uninfected 9HTEo- cells in vitro. Analogous to the in vivo model, expression of the human NKG2D ligand ULBP2 increased following 24 h of P. aeruginosa infection in vitro (Fig. 2).
Cells expressing NKG2D receptor are resident cells of the lung. NK cells, CD8+ T cells, and + T cells are known to express NKG2D in mice (12, 23). Lung digestion and flow cytometry were performed to assess the presence of lymphocytes bearing the NKG2D receptor in the lungs of nave mice. As determined with a rat monoclonal antibody specific for mouse NKG2D, 20% of the lymphocyte population obtained from the perfused lungs expressed NKG2D (Fig. 3A and B), indicating that a significant population of receptor-bearing cells resides in the lung. The majority of the NK1.1+ cells (82%), but only 12% of the CD8+ cells, coexpressed the NKG2D receptor in the lung (Fig. 3C and D). There were no significant differences in the distribution of NKG2D-positive cells in the lung 24 h after P. aeruginosa infection (not shown).
NKG2D receptor blockade inhibits bacterial clearance in the lung. To examine the function of the NKG2D receptor-ligand system in acute bacterial infection, we blocked NKG2D function in vivo by administration of the CX5 blocking antibody 16 h prior to P. aeruginosa infection. NKG2D blockade significantly impaired pulmonary clearance 24 h after P. aeruginosa infection (Fig. 4). Mice pretreated with the anti-NKG2D antibody had approximately 20-fold more bacilli in their lungs than control PAO1-infected mice had. These results directly implicate NKG2D receptor activation in the host response to P. aeruginosa because NKG2D cells were not depleted by antibody administration, as the frequencies of NK cells and CD8+ cells were not reduced compared to the frequencies in control mice inoculated with IgG (not shown).
Inhibition of bacterial clearance with NKG2D receptor blockade is independent of macrophage phagocytosis. Given the importance of AM phagocytosis in bacterial clearance, we next examined whether inhibition of AM phagocytosis accounted for the decreased bacterial clearance in mice given the NKG2D blocking antibody. The number of BAL-derived AM that had ingested bacteria was slightly greater in mice that received the NKG2D receptor blockade than in control mice at 2 h postinfection (Fig. 5). There were no differences in AM phagocytosis at 8 h or 24 h.
NKG2D receptor blockade inhibits pulmonary cytokine production, NO production, and epithelial cell damage. Following 24 h of P. aeruginosa infection, we examined the expression of pleiotropic inflammatory cytokines (interleukin-1 [IL-1], TNF-, and IFN-), leukocyte inflammation, epithelial cell shedding, and bacterial clearance from the lung. Anti-NKG2D pretreatment significantly decreased the pulmonary cytokine levels of IL-1, TNF-, and IFN- at 24 h postinfection (Fig. 6A to C). Similarly, NO levels, as estimated from the nitrite concentration, were reduced in the BAL fluid of mice that had been pretreated with anti-NKG2D antibody (Fig. 7). Reduced inflammatory cytokine levels were not accompanied by significant changes in the inflammatory cell profile in the BAL fluid (Fig. 8A). There was also no significant difference in the total leukocyte counts between treatments at 24 h (control, 2.68 x 106 ± 0.36 x 106 cells; anti-NKG2D treatment, 2.21 x 106 ± 0.26 x 106 cells). Although there were no differences in the leukocyte number and composition in the BAL fluid between treatments, there was a significant decrease in airway epithelial cell shedding in mice pretreated with anti-NKG2D compared to the shedding in control-treated mice. Approximately three times more epithelial cell damage occurred in the IgG-treated P. aeruginosa-infected mice, as determined by the number of epithelial cells recovered in the BAL fluid (Fig. 8B).
DISCUSSION
Although airway epithelial cells are the first line of defense against environmental pathogens, our knowledge of the role of these cells in signaling to resident mucosal immune cells is limited. Our results provide evidence which further establishes that epithelial cell-lymphocyte interactions mediate pulmonary cytokine production, epithelial cell integrity, and bacterial clearance. Specifically, our data demonstrate that the NKG2D receptor function is an important mechanism in the host defense against acute P. aeruginosa infection.
NKG2D ligand expression represents an intriguing link between the host encounter with a pathogen and subsequent activation of the immune system. The NKG2D receptor recognizes stressed cells through structurally related ligands induced on the cell surface (45). The NKG2D receptor was first detected on natural killer cells (52), and its function was subsequently determined to be an activating receptor in response to major histocompatibility complex class I-like molecules upregulated in innate and adaptive immune responses to cellular and tissue stress (3, 6, 51). Subsequent investigations detected NKG2D on NK T cells, CD8+ T cells, and T cells (45). Our finding that RAE-1 ligands are upregulated in the lung in response to P. aeruginosa infection is consistent with the role proposed for these molecules in tissue surveillance. Immunoblot and anti-RAE-1 immunohistochemistry analyses provide direct in vivo evidence that lung epithelial cells rapidly respond to pathogenic stimuli and are capable of directly activating the pulmonary immune system. Furthermore, incubation of human airway epithelial cells with P. aeruginosa directly stimulated induction of the human NKG2D ligand ULBP2 (Fig. 2). This is significant because it suggests that direct recognition of the pathogen, possibly via TLR binding, is sufficient to induce NKG2D ligands. Along these lines, various strains of E. coli induce expression of the NKG2D ligand MICA on Caco-2 human intestinal epithelial cells in vitro (48). In addition, Hamerman et al. recently demonstrated that RAE-1 transcript and cell surface protein induction occurs in peritoneal macrophages in response to TLR ligands, including lipopolysaccharide, zymosan, and double-stranded RNA (19).
This study provides the first demonstration that the NKG2D effector function is required for a competent host response against a pathogen in vivo. The cellular and molecular events that mediate the progression of bacterial infection via NKG2D activation are not entirely clear, but they may involve attenuated production of cytokines by NKG2D-expressing cells in the lung. A key feature of the NKG2D receptor-ligand system is that the function does not depend on an increase in the number of effector cells but does depend on the condition of the tissue and its capacity to stimulate resident immune cells. Significant populations of NKG2D-expressing cells (i.e., NK cells, CD8+ cells) reside in the pulmonary submucosa and parenchyma of unexposed mice (Fig. 3), and resident lymphocytes in the lung are prodigious sources of cytokines, including IFN-, TNF-, and IL-1 implicated in the host response to bacterial pathogens (5, 15, 18, 31, 32). There are conflicting data on the significance of proinflammatory cytokines in the context of pulmonary infection. Several studies have demonstrated that IFN- production by NK T cells is beneficial in the host response to microbial infections, including P. aeruginosa infections (14, 27, 29, 39). Furthermore, Wiemann et al. recently reported that systemic downregulation of NKG2D impairs the ability of NK cells and CD8+ T cells to produce IFN- in response to Listeria monocytogenes infection (50). In contrast, other investigators have shown that IFN- is dispensable for effective clearance of a P. aeruginosa infection (38, 47). We predict that the inhibition of cytokine production in mice that receive NKG2D blockade represents a proximal event in the subsequent cascade of cytokine production and inhibits macrophage and epithelial cell functions (e.g., direct microbicidal activity) in response to P. aeruginosa infection. The central role of activated macrophages and airway epithelium in the clearance of P. aeruginosa is supported by the fact that neutrophil accumulation is not affected in mice that receive NKG2D blocking antibody compared to neutrophil accumulation in control mice. The data presented here support the idea that the reduction in proinflammatory or activating cytokines may contribute to the immunosuppression observed in NKG2D antibody-treated mice. Furthermore, the inhibition of cytokine production may reflect a generalized depression in the lymphocyte-mediated antibacterial defense mechanisms. However, without conclusive evidence for a mechanism(s) responsible for the reduced clearance, the data should be interpreted cautiously.
Two key components of antibacterial defense in the lung, neutrophil accumulation and macrophage phagocytosis, are not affected by NKG2D blockade at 24 h and likely do not account for the mechanism by which NKG2D activation mediates the progression of bacterial growth. The lack of effects on inflammation are not totally surprising given that studies have shown that the cytokines are not necessary for increased inflammation in animal models (4, 15). The fact that phagocytosis is not impaired is somewhat surprising given that IFN- is a key mediator in the activation of alveolar macrophage functions and the elaboration of oxidative metabolites (8-10), which are compromised in anti-NKG2D-treated mice (Fig. 7). We speculate that additional components of the antibacterial defense repertoire of the lung may also be compromised as a consequence of NKG2D receptor blockade. These pathways include elaboration of antimicrobial peptides from the airway epithelium and the possibility that NKG2D-expressing cells may directly contribute to the antimicrobial shield in the lung. Human pulmonary epithelial cells are capable of producing -defensin antibacterial peptides in response to P. aeruginosa infection, as well as TNF- and IL-1 (20), and mice deficient in -defensin1 exhibit compromised clearance of several pulmonary pathogens (37). Furthermore, IL-1 and TNF- production by lipopolysaccharide-stimulated monocytes is sufficient to increase the expression of -defensin2 in human pulmonary epithelial cells (49). Another facet of NKG2D activation that remains to be explored is the potential production of antimicrobial peptides by T cells and NK cells in response to NKG2D ligand expression. T cells and NK cells are known to produce factors that are directly cytotoxic to bacteria (1, 34, 35, 42), but the relevance and significance of these mediators remain to be elucidated in our system.
There is conflicting evidence concerning the consequences of epithelial cell injury in the context of bacterial clearance. Our data demonstrate that increased epithelial cell death is associated with increased bacterial clearance and are in agreement with one of the known functions of the NKG2D receptor-ligand system (i.e., cytotoxic effects against ligand-bearing cells). In this regard, Grassme et al. demonstrated that bronchial cell apoptosis does not occur in P. aeruginosa-infected mice deficient in the CD95 cell death receptor, which is accompanied by increased mortality compared to the mortality of wild-type infected mice (16). This indicates that shedding of airway epithelial cells in control mice prevents bacterial dissemination and thus contributes to bacterial clearance. In contrast, airway epithelial cells in culture are relatively resistant to P. aeruginosa-induced apoptosis (44), and nominal apoptosis of airway epithelium was reported in septic patients (22). These studies and our study should be interpreted cautiously in this regard, because these reports provide data obtained with different strains of pathogens, different experimental designs, and different reagents used to detect apoptosis.
The present study revealed that the induction of RAE-1 ligands and subsequent immune activation through the NKG2D receptor are a significant component of the pulmonary host defense against acute P. aeruginosa infection. This pathway does not involve significant alterations in inflammation or AM phagocytosis, but it does regulate the elaboration of key cytokines involved in host defense, including IL-1, TNF-, and IFN-. We hypothesize that NKG2D activation is a key regulator of cytokine production by pulmonary T cells and NK cells and that inhibition of this production by NKG2D blockade leads to suppression of subsequent mediators of host defense. Further investigation of the mechanism of NKG2D receptor-ligand interactions in the lung should contribute to our understanding of the pulmonary innate immune system. Therapeutic strategies based solely on antibiotics remain limited due to the extensive ability of P. aeruginosa to develop antibiotic resistance, and novel strategies that enhance the host immune response against this pathogen may lead to reagents that improve the outcome of treatment of P. aeruginosa infections.
ACKNOWLEDGMENTS
We thank Michael P. Davis and Steve Ziegler for helpful discussions.
This study was supported by the NIH/NIEHS Center for Environmental Genetics (grant P30-ES06096-02).
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